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Biomedical subjects

R Schauer

Publications and source records attributed to R Schauer.

At least 73 records · Page 4Linked to original sources
PubMed1997

The prevention of anastomotic leakage after total gastrectomy with local decontamination. A prospective, randomized, double-blind, placebo-controlled multicenter trial.

OBJECTIVE: A prospective, randomized, double-blind, placebo controlled multicenter trial was undertaken in 205 patients treated with total gastrectomy for gastric malignancies to evaluate whether local antimicrobial measures reduce the incidence of esophagojejunal anastomotic leakage. SUMMARY BACKGROUND DATA: Anastomotic leakage of the esophagojejunostomy is always a septic complication of total gastrectomy for gastric malignancies, but it never has been attempted to prevent this complication with the administration of topical antimicrobial agents during the critical phase of anastomotic wound healing. METHODS: To evaluate the efficacy and safety of topical decontamination, the study was carried out as a prospective, randomized, double-blind and placebo-controlled clinical multicenter trial in patients with total gastrectomy for gastric cancer. Patients received either placebo or decontamination with polymyxin B (100 mg), tobramycin (80 mg), vancomycin (125 mg), and amphotericin B (500 mg) four times per day orally from the day before the operation until the seventh postoperative day. All patients received a perioperative intravenous prophylaxis with cefotaxime 2 x 2 g. Other interventions including the administration of antibiotics and fluids, were not affected by the study protocol. RESULTS: Of 260 patients who were randomized, total gastrectomy was not carried out in 55 patients. They dropped out of the study. Patients receiving an esophagojejunostomy were observed until day 42, when they were discharged from the clinic or died. An intention-to-treat analysis of the data was carried out. Among the 103 recipients of placebo, there were 11 (10.6%) with an anastomotic leakage of the esophagojejunostomy, and among the 102 recipients of decontamination, there were 3 (2.9%) with an anastomotic leakage of the esophagojejunostomy (p = 0.0492). Pulmonary infections were observed in 23 patients (22.3%) receiving placebo and in 9 patients (8.8%) who were decontaminated (p = 0.02). There were 11 deaths (10.6%) among the recipients of placebo and 5 deaths (4.9%) among the recipients of decontamination (p = 0.1). CONCLUSIONS: Decontamination with polymyxin, tobramycin, vancomycin, and amphotericin B during anastomotic wound healing is safe and effective in the prevention of esophagojejunal anastomotic leakage after total gastrectomy.

Aged↗
PubMed1997

[Dermatophytes do not produce sialidase in vitro].

Sialidase (EC 3.2.1.18) is a pathogenicity factor of many microorganisms, and may also play a role in adhesion of dermatophytes to the epithelia of their hosts by the hydrolytical cleavage of terminal, negatively charged sialic acids of glycoconjugates on the cell surfaces, thus allowing fungal lectins to bind to the subterminal sugars. Therefore, 116 strains of seven species of dermatophytes were investigated for sialidase production. Two highly sensitive, quantitative sialidase assays were applied to cell homogenates and culture supernatants from seven different media of the fungi, but were always negative for sialidase activity. However, sialidase activity was always detected in Ophiostoma stenoceras used as a positive control cultivated in parallel; the enzyme was inducible by sialylated mucins. A sialidase-dependent pathomechanism for dermatophytes appears unlikely based on the results presented.

Animals↗
PubMed1997

Lack of evidence for sialidase activity in Helicobacter pylori.

The role of sialic acid for the adhesion of Helicobacter pylori to gastric mucosa cells and/or to the mucin layer is still under debate. Several but not all H. pylori strains express a sialic acid-binding adhesin, specific for terminal alpha-2,3-sialic acid residues. Recently, the production of sialidase by H. pylori was reported [Dwarakanath, A.D. et al. (1995) FEMS Immunol. Med. Microbiol. 12,213 216]. We analysed several strains isolated from gastric biopsies cultivated both in liquid media and on agar plates for sialidase. Activity of this enzyme was first assayed using the fluorigenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid. Since the fluorimetric assay can give false-positive results caused by non-specific interactions with umbelliferyl-tagged substances, we used also the more sensitive and specific assay with sialyl-[3H]lactitol as a substrate. No evidence for sialidase activity of H. pylori strains, cultivated under both inducible and non-inducible conditions, was obtained.

Dyspepsia↗
PubMed1997

[Prognostic factors in gallbladder carcinoma as a decision aid for reoperation].

In a retrospective study of patients with gallbladder carcinoma, predictive factors for survival were evaluated. TNM stage, according to the UICC and complete tumor resection, turned out to be the strongest values in multivariate analysis. So it is important to make an early correct diagnosis and to reach complete tumor resection during surgical procedure. In case of an incidental tumor after simple cholecystectomy, hepatic wedge resection and lymph node dissection should be performed in T2 stage and above.

Adult↗
PubMed1996

CMP-N-acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron-sulphur protein to be described in Eukarya.

Electron paramagnetic resonance (EPR) spectroscopy and analysis of the primary structure of the CMP-N-acetylneuraminic acid hydroxylase revealed that this enzyme is the first iron-sulphur protein of the Rieske type to be found in the cytosol of Eukarya. The dithionite-reduced hydroxylase exhibited an EPR signal known to be characteristic for a Rieske iron-sulphur centre (2Fe-2S), the g-values being 1.78, 1.91 and 2.01, respectively. An analysis of the primary structure of the hydroxylase led to the identification of an amino acid sequence, known to be characteristic for Rieske proteins. Furthermore, possible binding sites for cytochrome b5, the substrate CMP-Neu5Ac and a mononuclear iron centre were also identified.

Amino Acid Sequence↗
PubMed1996

Localization of the putative sialic acid-binding site on the immunoglobulin superfamily cell-surface molecule CD22.

B-lymphocyte antigen CD22 is a member of the recently described sialoadhesin family of immunoglobulin-like cell-surface glycoproteins that bind glycoconjugates terminating in sialic acid. One prominent ligand for CD22 is the highly glycosylated leukocyte surface protein CD45. Using surface plasmon resonance spectroscopy, we characterized the interaction of recombinant mouse CD22 with native CD45 purified from rat thymus (CD45-thy). By in situ desialylation and resialylation of immobilized CD45-thy, we show that mouse CD22 binds to the sialoglycoconjugate NeuGc alpha 2-6Gal beta 1-4GlcNAc carried on CD45-thy N-glycans. Previous studies have shown that the sialic acid-binding site lies within the two membrane-distal domains of CD22 (domains 1 and 2), which are V-set and C2-set immunoglobulin superfamily domains, respectively. To further localize the binding site, we have made 42 single amino acid substitutions throughout both domains. All 12 mutations that abrogated binding to CD45-thy without disrupting antibody binding were of residues within the GFCC'C" beta-sheet of domain 1. These residues are predicted to form a contiguous binding site centered around an arginine residue in the F strand that is conserved in all members of the sialoadhesin family. Our results provide further evidence that immunoglobulin superfamily cell adhesion molecules use the GFCC'C" beta-sheet of membrane-distal V-set domains to bind structurally diverse ligands, suggesting that this surface is favored for cell-cell recognition.

Amino Acid Sequence↗
PubMed1996

Expression and purification of a recombinant "small" sialidase from Clostridium perfringens A99.

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.

Aminopeptidases↗
PubMed1996

Constitutively hyposialylated human T-lymphocyte clones in the Tn-syndrome: binding characteristics of plant and animal lectins.

Previously, beta 1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnher et al. (1992) Eur J Immunol 22: 1835-42]. Tn+ T lymphocytes express only Tn antigen GalNAc alpha 1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Ac alpha 2,6GalNAc alpha 1-O-R) or TF (Gal beta 1-3GalNAc alpha 1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins: Amaranthus caudatus (ACA), Maackia amurensis (MAA), Limax flavus (LFA), Sambucus nigra (SNA) and Triticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that the in vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.

Animals↗
PubMed1996

Binding specificity of influenza C-virus to variably O-acetylated glycoconjugates and its use for histochemical detection of N-acetyl-9-O-acetylneuraminic acid in mammalian tissues.

The specificity of influenza C-virus binding to sialoglycoconjugates was tested with various naturally O-acetylated gangliosides or synthetically O-acetylated sialic acid thioketosides, which revealed binding to 9-O-acetylated N-acetylneuraminic acid. Binding was also observed with a sample of Neu5,7Ac2-GD3, however at a lower degree. Sialic acids with two or three O-acetyl groups in the side chain of synthetic sialic acid derivatives are not recognized by the virus. In these experiments, bound viruses were detected with esterase substrates. Influenza C-virus was also used for the histological identification of mono-O-acetylated sialic acids in combination with an immunological visualization of the virus bound to thin-sections. The occurrence of these sialic acids was demonstrated in bovine submandibular gland, rat liver, human normal adult and fetal colon and diseased colon, as well as in human sweat gland. Submandibular gland and colon also contain significant amounts of glycoconjugates with two or three acetyl esters in the sialic acid side chain, demonstrating the value of the virus in discriminating between mono- and higher O-acetylation at the same site. The patterns of staining showed differences between healthy persons and patients with colon carcinoma, ulcerative colitis or Crohn's disease. Remarkably, some human colon samples did not show O-acetyl sialic acid-specific staining. The histochemical observations were controlled by chemical analysis of tissue sialic acids.

Acetylation↗
PubMed1996

The Sialoadhesins--a family of sialic acid-dependent cellular recognition molecules within the immunoglobulin superfamily.

For many years evidence has accumulated that sialic acids function in cellular interactions either by masking or as a recognition site. However, receptors or adhesion molecules mediating such functions between eukaryotic cells were unknown until about 5 years ago, when it was found that the members of the Selectin family mediate adhesion of leukocytes to specific endothelia through binding to sialylated glycans like sialyl Lewis. More recently, the Sialoadhesin family of sialic acid-dependent adhesion molecules was defined within the superfamily of immunoglobulin-like molecules. So far, it has been shown that sialoadhesin (Sn), CD22, CD33, the myelin-associated glycoprotein (MAG) and the Schwann cell myelin protein (SMP) belong to this family. In contrast to the Selectins, these proteins are associated with diverse biological processes, i.e. hemopoiesis, neuronal development and immunity. In this review their properties, carbohydrate specificities and potential biological functions are discussed. Finally, we provide perspectives with respect to the nature of ligands, implications of sialic acid modifications and future research.

Amino Acid Sequence↗
PubMed1996

Adhesion of Helicobacter pylori strains to alpha-2,3-linked sialic acids.

Helicobacter pylori is a human pathogen associated with gastritis and peptic ulcer. Adhesion properties of H. pylori to various structures have been described in the literature, including evidence for sialic acid-binding. To study the specificity and frequency of sialic acid-binding, fourteen H. pylori strains were investigated using haemagglutination with derivatized erythrocytes carrying sialic acids only on defined glycans and using haemagglutination inhibition assays. From these studies H. pylori strains can be grouped into sialic acid-dependent and sialic acid-independent classes. The sialic acid-dependent strains require alpha-2,3-linked sialic acid for haemagglutination. The potential roles of sialic acid-dependent adhesions for H. pylori-related infections are discussed.

Asialoglycoproteins↗
PubMed1996

Molecular dynamics-derived conformation and intramolecular interaction analysis of the N-acetyl-9-O-acetylneuraminic acid-containing ganglioside GD1a and NMR-based analysis of its binding to a human polyclonal immunoglobulin G fraction with selectivity for O-acetylated sialic acids.

The influence of 9-O-acetylation of GD1a, yielding GD1a (eNeu5,9Ac2) with a 9-O-acetylated sialic acid moiety linked to the outer galactose residue, on the spatial extension and mobility of the carbohydrate chain and on recognition by a natural human antibody is analysed. To study a potential impact of the O-acetyl group on the overall conformation of the carbohydrate chain, molecular dynamics (MD) simulations of oligosaccharide chain fragments of increasing length starting from the non-reducing end have been carried out for the first time in this study. They revealed a considerable loss in chain flexibility after addition of the internal N-acetylneuraminic acid onto the chain. Besides MD calculations with different dielectric constants, the conformational behaviour of the complete oligosaccharide chain of the 9-O-acetylated GD1a ganglioside was simulated in the solvents water and dimethyl sulfoxide. These solvents were also used in NMR measurements. The results of this study indicate that 9-O-acetylation at the terminal sialic acid does not influence the overall conformation of the ganglioside. An extended interaction analysis of energetically minimized conformations of GD1a (eNeu5,9Ac2) and GD1a, obtained during molecular dynamics simulations, allowed assessment of the influence of the different parts of the saccharide chains on spatial flexibility. Noteworthy energetic interactions, most interestingly between the 9-O-acetyl group and the pyranose ring of N-acetylgalactosamine, were ascertained by the calculations. However, the strength of this interaction does not force the ganglioside into a conformation, where the 9-O-acetyl group is no longer accessible. Binding of GD1a (eNeu5,9Ac2) to proteins, which are specific for 9-O-acetylated sialic acids, should thus at least partially be mediated by the presence of this group. To experimentally prove this assumption, a NMR study of 9-O-acetylated GD1a in the presence of an affinity-purified polyclonal IgG fraction from human serum with preferential binding to 9-O-acetylated sialic acid was performed. The almost complete disappearance of the intensity of the 9-O-acetyl methyl signal of the GD1a (eNeu5,9Ac2) clearly indicates that the assumed interaction of the 9-O-acetyl group with the human protein takes place.

Acetylation↗
PubMed1996

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity.

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.

Animals↗
PubMed1996

Isolation and characterization of sialate lyase from pig kidney.

Sialate lyase (sialate aldolase; systematic name N-acetylneuraminate pyruvate-lyase, EC 4.1.3.3) was isolated as soluble enzyme from pig kidney and purified 630-fold using a heating step, gel filtration, and chromatography on immobilized neuraminic acid beta-methyl glycoside in 14% yield to apparent homogeneity as tested by SDS-gel electrophoresis. The molecular mass is 58 kDa and the pH-optimum is at pH 7.2. Kinetic parameters were determined with N-acetyl-neuraminic acid as substrate: Km 3.7 mM and Vmax 37.1 mU. The lyase cleaves only free sialic acids with relative rates of 100% for N-acetylneuraminic acid, 55% for N-glycolylneuraminic acid and 32% for N-acetyl-9-O-acetylneuraminic acid, whereas N-acetyl-4-O-acetylneuraminic acid or 2-deoxy-2,3-didehydro-N-acetylneuraminic acid are not substrates. Enzyme activity was inhibited with p-chloromercuribenzoate, o-phenanthroline, cyanide, 5-diazonium-1-H-tetrazole, 5,5'-dithiobis(2-nitrobenzoic acid), diethylpyro-carbonate, and Rose Bengal in the presence of light and O2. Reduction with sodium borohydride in the presence of N-acetylneuraminic acid or pyruvate resulted in irreversible inhibition of enzyme activity. The inhibition experiments suggest the involvement of histidine, lysine and SH-residues in enzyme catalysis. Thus, this mammalian lyase most probably belongs to the Class I aldolases, and has properties similar to the same enzyme from Clostridium perfringens and is active with the alpha-form of N-acetylneuraminic acid.

Amino Acids↗
PubMed1996

[Experiences with liver transplantation and liver transplantation combined with Whipple's operation in Klatskin tumor].

In a retrospective Study on patients with type III/IV Klatskin tumors, results of liver transplantation and liver replacement in combination with a Whipple procedure and extensive lymph node dissection (six and four patients, respectively) were compared. The combined ultraradical approach was not associated with higher peri- and postoperative morbidity and mortality, and seems to offer a chance for better results in terms of tumor-free survival.

Adult↗
PubMed1995

Distribution of developmentally regulated trans-sialidases in the Kinetoplastida and characterization of a shed trans-sialidase activity from procyclic Trypanosoma congolense.

The expression of developmentally regulated sialidase and trans-sialidase activities in kinetoplastid protozoa was investigated. The occurrence of these enzymes was found not to be a common feature among the Kinetoplastida, but to be restricted to distinct developmental life cycle stages of only a few species. While sialidases without trans-sialylating activities were demonstrated in Trypanosoma vivax and T. rangeli, trans-sialidase activity is expressed throughout the brucei-group and in T. congolense. Neither T. evansi, nor T. equiperdum express sialidases or trans-sialidases. Furthermore, the absence of both, sialidase and trans-sialidase activities was proven in the Leishmania, Crithidia, Herpetomonas, Leptomonas and Phytomonas, respectively. In all species tested, the occurrence of sialic acids coincides with the expression of trans-sialidase activity. Those parasites, which lack trans-sialidases or only display regular sialidases, also lack cell-bound sialic acids. The regular sialidase activity from bloodstream form T. vivax was characterized. The trans-sialidase from T. congolense is restricted to the procyclic culture forms and is shed into the culture medium. The enzyme has a pH-optimum at pH 7.0, displays sensitivity towards chlorides and is resistant against commonly used sialidase inhibitors. T. congolense trans-sialidase transfers preferentially alpha(2-3)-linked sialic acids onto terminal beta-galactose residues. Also hydroxylated sialic acids (Neu5Gc) are transferred. The major glycoprotein GARP from procyclic T. congolense was identified as one potential natural sialic acid acceptor on the parasite's surface. In order to facilitate the characterization of trans-sialidases a novel, fluorimetric trans-sialidase assay was developed.

Animals↗